The endoplasmic reticulum (ER) is comprised of two major domains, the nuclear envelope and peripheral ER. The peripheral ER has a complex morphology, consisting of interconnected ER tubules and membrane sheets, and is remarkably dynamic; its ER tubules, often highly mobile along the cytoskeleton, have been implicated in a wide range of processes including mitochondrial division and autophagosome formation. The reticulon family, comprised of four paralogs in mammalian cells, is a central contributor toward the elaborate architecture of the peripheral ER. Reticulons are thought to create and maintain ER regions of high membrane curvature, including ER tubules and membrane sheet edges. Although multiple lines of evidence support this vital role for reticulons in ER membrane shape, the detailed contributions of individual reticulon family members remain largely unclear. As an initial step towards delineating the roles that individual reticulons have in ER morphology, we will focus on Reticulon-3 (Rtn3), which is ubiquitously expressed and implicated in health relevant processes such as apoptosis, viral replication, and neurodegenerative diseases. This proposed research training program will, (1) examine the precise relationship between ER structure and Rtn3 localization using cryo-electron tomography, allowing us to visualize metallothionein-tagged Rtn3 on a 3-dimensional structure of the ER; (2) characterize the functional role of Rtn3 by examining the effects of Rtn3 depletion on ER structure with cryo-electron tomography, and also identifying whether Rtn3 specifically localizes to dynamic (i.e. mobile) ER tubules or those associated with organelles; (3) determine whether Rtn3 function is regulated, particularly, in a cell cycle dependent manner by identifying post-translational protein modifications and changes in Rtn3 levels throughout the cell cycle. This proposed research training program will greatly enhance our understanding of the functional role of Rtn3 and acts as a first step towards individually characterizing the contributions of reticulons to ER architecture.